Fibroblast Growth Factors and their Receptors
Fibroblast growth factors (FGFs) comprise a large family of evolutionarily conserved polypeptides involved in a variety of biological processes including morphogenesis, angiogenesis, and tissue remodeling as well as in the pathogenesis of numerous diseases. The various members of this family stimulate the proliferation of a wide spectrum of cells, including those deriving from mesenchymal, endothelial, epithelial and neuroectodermal origin. FGFs are expressed in a strict temporal and spatial pattern during development and have important roles in patterning and limb formation (reviewed in Ornitz, 2000). All members of the FGF family share a homology core domain of about 120 amino acids, 28 aa residues are highly conserved and four are identical. The adjacent N- and C-termini are of variable length and share limited homology. The core domain comprises both the primary receptor binding sites and a heparin-binding domain, which are distinct from each other (reviewed in Ornitz and Itoh, 2001).
Fibroblast growth factor 2, also known as FGF2, basic FGF, bFGF, prostatin and heparin binding growth factor 2, is highly conserved among species and has been shown to stimulate the proliferation of a wide variety of cell types. Human FGF2 is expressed in several forms, a 210 aa precursor, a 155 aa form, a 146 aa N-terminal truncated form and several others (reviewed in Okada-Ban et al., 2000). A method for purifying recombinant FGF2 has been disclosed in WO 91/09126.
The biological response of cells to FGF is mediated through specific, high affinity (Kd 20-500 pM) cell surface receptors that possess intrinsic tyrosine kinase activity and are phosphorylated upon binding of FGF. Five distinct Fibroblast Growth Factor Receptors (FGFRs) have been identified, FGFR1-4 are transmembrane-protein kinases while FGFR5 lacks a tyrosine kinase domain. The FGFR extracellular domain consists of three immunoglobulin-like (Ig-like) domains (D1, D2 and D3), a heparin binding domain and an acidic box. Alternative splicing of D3 in FGFR1-3 mRNAs generates six different receptor subtypes, each having unique ligand specificity and tissue distribution pattern.
Another critically functional component in receptor activation is the binding to soluble heparin or a heparan sulfate proteoglycan. Different models have been proposed to explain the role of heparan sulfate proteoglycans (HSPG) in FGF signaling, including the formation of a functional tertiary complex between FGF, FGFR and an HSPG (Yayon et al., 1991). Most naturally occurring heparan sulfate are incapable of promoting heparin dependent high affinity FGF receptor binding and activation (Aviezer et al., 1994). Moreover, heparan sulfate which is locally secreted by cells, masks receptor specificity of the FGF ligands.
FGFRs and Disease
A number of birth defects affecting the skeleton are associated with mutations in the genes encoding FGF receptors. Certain FGFRs have been implicated in certain malignancies and proliferative diseases. FGFR3 is the most frequently mutated oncogene in transitional cell carcinoma (TCC) of the bladder where it is mutated in about 50% of the cases; the FGFR3IIIc isoform is ectopically expressed in 15-20% of patients with multiple myeloma and is over expressed in the white blood cells of chronic myeloid leukemia (CML) patients. A mutation in FGFR3 is linked to cervical carcinoma. FGFR4 was shown to be associated with pituitary tumors and breast cancer progression. In contrast, certain FGF ligands have been shown to be highly useful for treating indications including wounds (U.S. Pat. Nos. 4,950,483, 5,859,208 and 6,294,359), myocardial infarction (U.S. Pat. Nos. 4,296,100 and 4,378,347), skeletal disorders (U.S. Pat. Nos. 5,614,496 and 5,656,598) and for remodeling cardiac tissue (U.S. Pat. No. 6,352,971).
FGF Variants and Receptor Specificity
All members of the FGF family share a homology core domain of about 120 amino acids (aa), 28 aa residues are highly conserved and four are identical. Structural studies on several FGFs identified twelve antiparallel β strands each one adjacent to β-loops comprising the core region, conserved throughout the family. The core domain comprises the primary FGFR and heparin binding sites. Receptor binding regions are distinct from heparin binding regions (reviewed in Ornitz and Itoh, 2001).
In view of the large number of FGFs and FGF receptor variants, a major question regarding FGF function is their receptor specificity or selectivity. Most FGF ligands bind more than one receptor subtype and such a degree of cross-reactivity is shared between all FGF receptors, demonstrating a highly redundant signaling network. All FGFRs tested so far bind FGF1 (acidic FGF, aFGF) with moderate to high affinity, further demonstrating the apparent redundancy in the FGF system (Ornitz et al., 1996).
Various types of FGF variants are known in the art. U.S. Pat. No. 6,294,359 discloses agonist and antagonist analogs of FGF2 that comprise amino acid substitutions in the C8 and C96 residues. U.S. Pat. No. 5,352,589 discloses derivatives in the C78 and C96 residues. U.S. Pat. No. 5,352,589 discloses derivatives of FGF2 that act as antagonists or speragonists. On particular construct comprises a human or boine derivatie wherein amino acids 27-32 have been deleted. Wong et al., (1995) identified putative heparin binding domains in FGF1 (154aa form) based on consensus sequence motifs, including amino acids 22-27. Nevertheless, according to that citation substitution of Lys23, Lys24 or Lys26 with glycine residues had no effect on the activity of FGF1.
Attempts have been made to achieve altered FGF receptor specificity by mutating or truncating the ligands, by means of mutations introduced at certain locations within the gene encoding for the proteins. Certain truncated and mutated variants have been disclosed by some of the inventors of the present invention in PCT publications WO 02/36732 and WO 03/094835. International patent applications WO 02/36732 and WO 03/094835 of some of the applicants of the present invention, disclose FGF variants having at least one amino acid substitution in the β8-β9 loop, and/or an N- and/or C-terminal truncation, having increased receptor specificity to one receptor subtype compared to the corresponding wild type FGF. PCT publication WO 02/36732 discloses specific FGF9 variants having 36, 44 or 63 amino acid truncations at the N-terminus. The shortest variant was shown to retain weak activity towards FGFR3 while losing almost all activity towards FGFR1. PCT publication WO 03/094835 teaches an FGF4 variant having both an N-terminal truncation (55 amino acids) and an amino acid substitution in the β8-β9 loop, the variant exhibiting enhanced receptor specificity towards FGFR3 with substantially unchanged activity towards FGFR1 and FGFR2.
Several investigators have demonstrated FGF mutants and variants affecting receptor and heparin binding. Kuroda et al., (1999) demonstrated that a full-length FGF4 polypeptide (191 aa) and an N-terminal truncated version containing 134 amino acid residues exhibit comparable cellular proliferation on NIH3T3 cells and increase of bone density. The shortest form of FGF4 tested, containing only 111 amino acid residues, exhibited limited growth stimulatory activity.
U.S. Pat. No. 5,998,170 discloses a biologically active FGF16 molecule having from one to thirty-four amino acids deleted from the N-terminus or from one to eighteen amino acids deleted from the C-terminus. The truncated ligands were shown to retain biological activity including hepatocellular proliferation and increased production of triglycerides and serum proteins, when administered to animals.
X-ray crystallography has been used in an attempt to study the basis of specificity of FGFs to their receptors (Plotnikov et al., 2000; Olsen, et al., 2004; Mohammadi et al., 2005). The role of the N-terminal domain of the FGFs was resolved in only a few of the abovementioned crystal structures. Olsen et al., (2004) compared receptor binding of a full length FGF1 (155 aa) to a N-terminal truncated form (21-155) and show that the N-terminus of FGF1 may be relevant to binding and activation of the FGFR3c isoform. The (21-155) form also exhibits reduced FGFR2 and FGFR3 phosphorylation.
Plotnikov et al., (2000) determined the crystal structures of FGF1 and FGF2 complexed with the ligand binding domains (Ig-like domains 2 and 3) of FGFR1 and FGFR2, respectively and shows that certain N-terminal residues of FGF, in particular Phe17 and Lys18 of FGF2 (Lys27 of 155 aa form), could be in contact with the D3 domain of FGFR2. The authors speculated, but did not provide experimental evidence, that amino acids 7-13 of FGF1 play a role in receptor binding. The deletion of that specific sequence, which had originally been proposed to be a nuclear localization sequence, reduces the ability of FGF1 to induce cell proliferation in endothelial cell lines by about 250-fold (Imamura et al., 1990). These findings neither suggest nor teach that the deletion of that specific sequence would affect receptor selectivity.
Seno et al., (1990) teach certain bFGF variants having N- and C-terminus truncations and have characterized their ability to bind heparin. The mitogenic activity of those variants in BALB/c3T3 cells was determined and the N14 variant (corresponding to a 22 amino acid truncation of the 155 aa bFGF species) showed an activity of 68% that of the mature form of bFGF. A much larger truncation, N41, which corresponds to a 49 amino acid truncation of the 155 aa species, exhibits only about 2% mitogenic activity. There is neither teaching nor suggestion of a truncated FGF exhibiting receptor selectivity toward one or another FGFR species.
In another study the basic residues in an analogous stretch of FGF2 (aa 27-31, 155 aa form) were modified to neutral glutamine residues, specifically K27Q, K30Q and R31Q (Presta et al., 1993). That mutant retains receptor binding capacity and mitogenic activity on endothelial cells yet exerts reduced uPA inducing activity.
The above disclosures show that modifications in certain N-terminal residues affect cell proliferation, yet they neither teach nor suggest that mutations or substitutions in N-terminal residues of FGF would affect receptor selectivity.
A hexapeptide, consisting of N-terminal amino acids 13-18 of FGF2 (146 aa form, corresponding to aa 22-27 of the 155 aa form), was shown to inhibit binding of FGF2 to FGFR-1, implicating this motif in receptor binding (Yayon et al, 1993). There was neither teaching nor suggestion of receptor specificity.
Attempts have been made to alter FGF receptor specificity and heparin binding by means of site directed mutagenesis within the FGF genes. U.S. Pat. No. 5,512,460 discloses a biologically active FGF9 (glia activating factor, GAF) molecule comprising N-terminus and C-terminus truncations of 53 aa and 13 aa, respectively. U.S. Pat. No. 5,571,895 discloses an N-terminus 54 aa deletion yielding a 154 aa protein retaining its biological activity, as measured by glial cell growth activity.
U.S. Pat. No. 5,491,220 to one of the inventors of the present application discloses structural analogues of FGF2 comprising substitution of the β9-β10 loop having altered biological properties and binding specificity.
Springer et al., (1994) identified two FGFR binding sites on FGF2, the first includes hydrophobic residues Y24, Y103, L140 and M142 and polar residues R44, and N101. The author concludes that R44 and N101 were the only polar residues observed to be important in the primary binding interaction between FGF2 and FGFR.
In an attempt to identify FGF2 antagonists having reduced binding affinity towards FGFR1. Zhu et al. (1997) tested N101A, N102A, Y103A, N104A and T105A muteins and their binding affinity to FGFR1. These correspond to amino acid residues N110, N111, Y112, N113 and T114 of the 155 aa species. The N101A and N102A muteins were shown to have FGFR1 binding similar to that of the wild type protein, while the N104A mutein exhibited 400 fold reduced FGFR1 binding.
There is neither teaching nor suggestion of receptor selectivity in the above-cited references.
Bellosta et al., (2001) have disclosed mutated and truncated FGF4 variants having reduced receptor binding and a low mitogenic potential. Of interest is a truncated variant, which lacks 78 N-terminal amino acids and, according to the published data, retains the core domain, exhibiting FGFR binding affinities similar to that of the wild type ligand. Certain mutations within the core domain were shown to have a deleterious effect on both DNA synthesis and receptor binding.
The extensive efforts made to produce truncation, deletion and point mutation variants in FGF have resulted in certain alterations in receptor specificity. There remains an unmet need for highly active and selective ligands for the various FGF receptor isoforms, useful in selective stimulation or inhibition of these receptors, thereby addressing the clinical manifestations associated with receptor mutations, and modulation of various biological functions.
It is to be understood that known variants of FGF are excluded explicitly from the present invention.
A need for FGF variants having increased receptor selectivity is manifest. Lack of receptor selectivity is often detrimental to tissue repair and regeneration both ex vivo and in vivo. For instance, FGFR1 activation is often critical for cell survival and proliferation; hence, a ligand having enhanced FGFR1 specificity would be ideal for supporting physiological FGF mediated activities in processes such as wound healing, independent of the heparan sulfate environment.